Project Summary Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive co- transcriptional processing in the nucleus before they can be exported to the cytoplasm and function as mRNAs. The processing events include 5??-end capping, splicing, and 3??- end cleavage and polyadenylation. The 3'-end processing of most pre-mRNAs requires a large number of protein factors for its execution, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II, and poly(A) polymerase (PAP). The 3'-end processing machinery in yeast has similarity to that in mammals, although there are also significant differences. Replication-dependent histone pre-mRNAs contain a conserved stem-loop near their 3??- end and employ a distinct machinery for its processing, although it shares some protein factors with the canonical pre-mRNA 3??-end processing machinery. mRNA 5??-end capping occurs early during transcription by RNA polymerase II, and it was generally believed that capping always proceeds to completion. We have recently discovered that the Rai1/DXO family of proteins are part of a mRNA capping quality surveillance mechanism. They can possess RNA 5??-end pyrophosphohydrolase (PPH) and decapping activities, and help remove incompletely capped mRNAs from cells. Despite the extensive studies on these mRNA processing and quality control factors, significant gaps remain in our knowledge on their molecular mechanisms of action. We will carry out structural studies on the protein factors and their complexes, and assess the structural observations by careful biochemical and functional experiments. The proposed project will greatly enhance our understanding of these important events in mRNA lifecycle.